MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

Slow myosin heavy chain 1 is required for slow myofibril and muscle fibre growth but not for myofibril initiation.

Slow myosin heavy chain 1 (Smyhc1) is the major sarcomeric myosin driving early contraction by slow skeletal muscle fibres in zebrafish. New mutant alleles lacking a functional smyhc1 gene move poorly, but recover motility as the later-formed fast muscle fibres of the segmental myotomes mature, and are adult viable. By motility analysis and inhibiting fast muscle contraction pharmacologically, we show that a slow muscle motility defect persists in mutants until about 1 month of age. Breeding onto a genetic background marking slow muscle fibres with EGFP revealed that mutant slow fibres undergo terminal differentiation, migration and fibre formation indistinguishable from wild type but fail to generate large myofibrils and maintain cellular orientation and attachments. In mutants, initial myofibrillar structures with 1.67 â€‹µm periodic actin bands fail to mature into the 1.96 â€‹µm sarcomeres observed in wild type, despite the presence of alternative myosin heavy chain molecules. The poorly-contractile mutant slow muscle cells generate numerous cytoplasmic organelles, but fail to grow and bundle myofibrils or to increase in cytoplasmic volume despite passive movements imposed by fast muscle. The data show that both slow myofibril maturation and cellular volume increase depend on the function of a specific myosin isoform and suggest that appropriate force production regulates muscle fibre growth.

Isolation, Culture, and Analysis of Zebrafish Myofibers and Associated Muscle Stem Cells to Explore Adult Skeletal Myogenesis.

Adult skeletal musculature experiences continuous physical stress, and hence requires maintenance and repair to ensure its continued efficient functioning. The population of resident muscle stem cells (MuSCs), termed satellite cells, resides beneath the basal lamina of adult myofibers, contributing to both muscle hypertrophy and regeneration. Upon exposure to activating stimuli, MuSCs proliferate to generate new myoblasts that differentiate and fuse to regenerate or grow myofibers. Moreover, many teleost fish undergo continuous growth throughout life, requiring continual nuclear recruitment from MuSCs to initiate and grow new fibers, a process that contrasts with the determinate growth observed in most amniotes. In this chapter, we describe a method for the isolation, culture, and immunolabeling of adult zebrafish myofibers that permits examination of both myofiber characteristics ex vivo and the MuSC myogenic program in vitro. Morphometric analysis of isolated myofibers is suitable to assess differences among slow and fast muscles or to investigate cellular features such as sarcomeres and neuromuscular junctions. Immunostaining for Pax7, a canonical stemness marker, identifies MuSCs on isolated myofibers for study. Furthermore, the plating of viable myofibers allows MuSC activation and expansion and downstream analysis of their proliferative and differentiative dynamics, thus providing a suitable, parallel alternative to amniote models for the study of vertebrate myogenesis.

Skeletal Muscle Regeneration in Zebrafish.

Muscle regeneration models have revealed mechanisms of inflammation, wound clearance, and stem cell-directed repair of damage, thereby informing therapy. Whereas studies of muscle repair are most advanced in rodents, the zebrafish is emerging as an additional model organism with genetic and optical advantages. Various muscle wounding protocols (both chemical and physical) have been published. Here we describe simple, cheap, precise, adaptable, and effective wounding protocols and analysis methods for two stages of a larval zebrafish skeletal muscle regeneration model. We show examples of how muscle damage, ingression of muscle stem cells, immune cells, and regeneration of fibers can be monitored over an extended timecourse in individual larvae. Such analyses have the potential to greatly enhance understanding, by reducing the need to average regeneration responses across individuals subjected to an unavoidably variable wound stimulus.

Clonal behaviour of myogenic precursor cells throughout the vertebrate lifespan.

To address questions of stem cell diversity during skeletal myogenesis, a Brainbow-like genetic cell lineage tracing method, dubbed Musclebow2, was derived by enhancer trapping in zebrafish. It is shown that, after initial formation of the primary myotome, at least 15 muscle precursor cells (mpcs) seed each somite, where they proliferate but contribute little to muscle growth prior to hatching. Thereafter, dermomyotome-derived mpc clones rapidly expand while some progeny undergo terminal differentiation, leading to stochastic clonal drift within the mpc pool. No evidence of cell-lineage-based clonal fate diversity was obtained. Neither fibre nor mpc death was observed in uninjured animals. Individual marked muscle fibres persist across much of the lifespan indicating low rates of nuclear turnover. In adulthood, early-marked mpc clones label stable blocks of tissue comprising a significant fraction of either epaxial or hypaxial somite. Fusion of cells from separate early-marked clones occurs in regions of clone overlap. Wounds are regenerated from several local mpcs; no evidence for specialised stem mpcs was obtained. In conclusion, our data indicate that most mpcs in muscle tissue contribute to local growth and repair and suggest that cellular turnover is low in the absence of trauma.

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Cellular and molecular pathways controlling muscle size in response to exercise.

From the discovery of ATP and motor proteins to synaptic neurotransmitters and growth factor control of cell differentiation, skeletal muscle has provided an extreme model system in which to understand aspects of tissue function. Muscle is one of the few tissues that can undergo both increase and decrease in size during everyday life. Muscle size depends on its contractile activity, but the precise cellular and molecular pathway(s) by which the activity stimulus influences muscle size and strength remain unclear. Four correlates of muscle contraction could, in theory, regulate muscle growth: nerve-derived signals, cytoplasmic calcium dynamics, the rate of ATP consumption and physical force. Here, we summarise the evidence for and against each stimulus and what is known or remains unclear concerning their molecular signal transduction pathways and cellular effects. Skeletal muscle can grow in three ways, by generation of new syncytial fibres, addition of nuclei from muscle stem cells to existing fibres or increase in cytoplasmic volume/nucleus. Evidence suggests the latter two processes contribute to exercise-induced growth. Fibre growth requires increase in sarcolemmal surface area and cytoplasmic volume at different rates. It has long been known that high-force exercise is a particularly effective growth stimulus, but how this stimulus is sensed and drives coordinated growth that is appropriately scaled across organelles remains a mystery.

Isolation of Myofibres and Culture of Muscle Stem Cells from Adult Zebrafish.

Skeletal muscles generate force throughout life and require maintenance and repair to ensure efficiency. The population of resident muscle stem cells (MuSCs), termed satellite cells, dwells beneath the basal lamina of adult myofibres and contributes to both muscle growth and regeneration. Upon exposure to activating signals, MuSCs proliferate to generate myoblasts that differentiate and fuse to grow or regenerate myofibres. This myogenic progression resembles aspects of muscle formation and development during embryogenesis. Therefore, the study of MuSCs and their associated myofibres permits the exploration of muscle stem cell biology, including the cellular and molecular mechanisms underlying muscle formation, maintenance and repair. As most aspects of MuSC biology have been described in rodents, their relevance to other species, including humans, is unclear and would benefit from comparison to an alternative vertebrate system. Here, we describe a procedure for the isolation and immunolabelling or culture of adult zebrafish myofibres that allows examination of both myofibre characteristics and MuSC biology ex vivo. Isolated myofibres can be analysed for morphometric characteristics such as the myofibre volume and myonuclear domain to assess the dynamics of muscle growth. Immunolabelling for canonical stemness markers or reporter transgenes identifies MuSCs on isolated myofibres for cellular/molecular studies. Furthermore, viable myofibres can be plated, allowing MuSC myogenesis and analysis of proliferative and differentiative dynamics in primary progenitor cells. In conclusion, we provide a comparative system to amniote models for the study of vertebrate myogenesis, which will reveal fundamental genetic and cellular mechanisms of MuSC biology and inform aquaculture. Graphic abstract: Schematic of Myofibre Isolation and Culture of Muscle Stem Cells from Adult Zebrafish.

mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation.

BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation. METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells. RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta. CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

Knockout of zebrafish desmin genes does not cause skeletal muscle degeneration but alters calcium flux.

Desmin is a muscle-specific intermediate filament protein that has fundamental role in muscle structure and force transmission. Whereas human desmin protein is encoded by a single gene, two desmin paralogs (desma and desmb) exist in zebrafish. Desma and desmb show differential spatiotemporal expression during zebrafish embryonic and larval development, being similarly expressed in skeletal muscle until hatching, after which expression of desmb shifts to gut smooth muscle. We generated knockout (KO) mutant lines carrying loss-of-function mutations for each gene by using CRISPR/Cas9. Mutants are viable and fertile, and lack obvious skeletal muscle, heart or intestinal defects. In contrast to morphants, knockout of each gene did not cause any overt muscular phenotype, but did alter calcium flux in myofibres. These results point to a possible compensation mechanism in these mutant lines generated by targeting nonsense mutations to the first coding exon.

Mef2c factors are required for early but not late addition of cardiomyocytes to the ventricle.

During heart formation, the heart grows and undergoes dramatic morphogenesis to achieve efficient embryonic function. Both in fish and amniotes, much of the growth occurring after initial heart tube formation arises from second heart field (SHF)-derived progenitor cell addition to the arterial pole, allowing chamber formation. In zebrafish, this process has been extensively studied during embryonic life, but it is unclear how larval cardiac growth occurs beyond 3 days post-fertilisation (dpf). By quantifying zebrafish myocardial growth using live imaging of GFP-labelled myocardium we show that the heart grows extensively between 3 and 5 dpf. Using methods to assess cell division, cellular development timing assay and Kaede photoconversion, we demonstrate that proliferation, CM addition, and hypertrophy contribute to ventricle growth. Mechanistically, we show that reduction in Mef2c activity (mef2ca+/-;mef2cb-/-), downstream or in parallel with Nkx2.5 and upstream of Ltbp3, prevents some CM addition and differentiation, resulting in a significantly smaller ventricle by 3 dpf. After 3 dpf, however, CM addition in mef2ca+/-;mef2cb-/- mutants recovers to a normal pace, and the heart size gap between mutants and their siblings diminishes into adulthood. Thus, as in mice, there is an early time window when SHF contribution to the myocardium is particularly sensitive to loss of Mef2c activity.

Circadian regulation of muscle growth independent of locomotor activity.

Muscle tissue shows diurnal variations in function, physiology, and metabolism. Whether such variations are dependent on the circadian clock per se or are secondary to circadian differences in physical activity and feeding pattern is unclear. By measuring muscle growth over 12-h periods in live prefeeding larval zebrafish, we show that muscle grows more during day than night. Expression of dominant negative CLOCK (ΔCLK), which inhibits molecular clock function, ablates circadian differences and reduces muscle growth. Inhibition of muscle contraction reduces growth in both day and night, but does not ablate the day/night difference. The circadian clock and physical activity are both required to promote higher muscle protein synthesis during the day compared to night, whereas markers of protein degradation, murf messenger RNAs, are higher at night. Proteasomal inhibitors increase muscle growth at night, irrespective of physical activity, but have no effect during the day. Although physical activity enhances TORC1 activity, and the TORC1 inhibitor rapamycin inhibits clock-driven daytime growth, no effect on muscle growth at night was detected. Importantly, day/night differences in 1) muscle growth, 2) protein synthesis, and 3) murf expression all persist in entrained larvae under free-running constant conditions, indicating circadian drive. Removal of circadian input by exposure to either permanent darkness or light leads to suboptimal muscle growth. We conclude that diurnal variations in muscle growth and metabolism are a circadian property that is independent of, but augmented by, physical activity, at least during development.

Myogenin is an essential regulator of adult myofibre growth and muscle stem cell homeostasis.

Growth and maintenance of skeletal muscle fibres depend on coordinated activation and return to quiescence of resident muscle stem cells (MuSCs). The transcription factor Myogenin (Myog) regulates myocyte fusion during development, but its role in adult myogenesis remains unclear. In contrast to mice, myog-/-zebrafish are viable, but have hypotrophic muscles. By isolating adult myofibres with associated MuSCs, we found that myog-/- myofibres have severely reduced nuclear number, but increased myonuclear domain size. Expression of fusogenic genes is decreased, Pax7 upregulated, MuSCs are fivefold more numerous and mis-positioned throughout the length of myog-/-myofibres instead of localising at myofibre ends as in wild-type. Loss of Myog dysregulates mTORC1 signalling, resulting in an 'alerted' state of MuSCs, which display precocious activation and faster cell cycle entry ex vivo, concomitant with myod upregulation. Thus, beyond controlling myocyte fusion, Myog influences the MuSC:niche relationship, demonstrating a multi-level contribution to muscle homeostasis throughout life.

Fgf-driven Tbx protein activities directly induce myf5 and myod to initiate zebrafish myogenesis.

Skeletal muscle derives from dorsal mesoderm formed during vertebrate gastrulation. Fibroblast growth factor (Fgf) signalling cooperates with Tbx transcription factors to promote dorsal mesoderm formation, but their role in myogenesis has been unclear. Using zebrafish, we show that dorsally derived Fgf signals act through Tbx16 and Tbxta to induce slow and fast trunk muscle precursors at distinct dorsoventral positions. Tbx16 binds to and directly activates the myf5 and myod genes, which are required for commitment to myogenesis. Tbx16 activity depends on Fgf signalling from the organiser. In contrast, Tbxta is not required for myf5 expression, but binds a specific site upstream of myod that is not bound by Tbx16 and drives (dependent on Fgf signals) myod expression in adaxial slow precursors, thereby initiating trunk myogenesis. After gastrulation, when similar muscle cell populations in the post-anal tail are generated from tailbud, declining Fgf signalling is less effective at initiating adaxial myogenesis, which is instead initiated by Hedgehog signalling from the notochord. Our findings suggest a hypothesis for ancestral vertebrate trunk myogenic patterning and how it was co-opted during tail evolution to generate similar muscle by new mechanisms.This article has an associated 'The people behind the papers' interview.

Maternal Larp6 controls oocyte development, chorion formation and elevation.

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.

EGF receptor (EGFR) inhibition promotes a slow-twitch oxidative, over a fast-twitch, muscle phenotype.

A low quadriceps slow-twitch (ST), oxidative (relative to fast-twitch) fiber proportion is prevalent in chronic diseases such Chronic Obstructive Pulmonary Disease (COPD) and is associated with exercise limitation and poor outcomes. Benefits of an increased ST fiber proportion are demonstrated in genetically modified animals. Pathway analysis of published data of differentially expressed genes in mouse ST and FT fibers, mining of our microarray data and a qPCR analysis of quadriceps specimens from COPD patients and controls were performed. ST markers were quantified in C2C12 myotubes with EGF-neutralizing antibody, EGFR inhibitor or an EGFR-silencing RNA added. A zebrafish egfra mutant was generated by genome editing and ST fibers counted. EGF signaling was (negatively) associated with the ST muscle phenotype in mice and humans, and muscle EGF transcript levels were raised in COPD. In C2C12 myotubes, EGFR inhibition/silencing increased ST, including mitochondrial, markers. In zebrafish, egfra depletion increased ST fibers and mitochondrial content. EGF is negatively associated with ST muscle phenotype in mice, healthy humans and COPD patients. EGFR blockade promotes the ST phenotype in myotubes and zebrafish embryos. EGF signaling suppresses the ST phenotype, therefore EGFR inhibitors may be potential treatments for COPD-related muscle ST fiber loss.

Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.

Tendons are an essential part of the musculoskeletal system, connecting muscle and skeletal elements to enable force generation. The transcription factor scleraxis marks vertebrate tendons from early specification. Scleraxis-null mice are viable and have a range of tendon and bone defects in the trunk and limbs but no described cranial phenotype. We report the expression of zebrafish scleraxis orthologs: scleraxis homolog (scx)-a and scxb in cranial and intramuscular tendons and in other skeletal elements. Single mutants for either scxa or scxb, generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), are viable and fertile as adult fish. Although scxb mutants show no obvious phenotype, scxa mutant embryos have defects in cranial tendon maturation and muscle misalignment. Mutation of both scleraxis genes results in more severe defects in cranial tendon differentiation, muscle and cartilage dysmorphogenesis and paralysis, and lethality by 2-5 wk, which indicates an essential function of scleraxis for craniofacial development. At juvenile and adult stages, ribs in scxa mutants fail to mineralize and/or are small and heavily fractured. Scxa mutants also have smaller muscle volume, abnormal swim movement, and defects in bone growth and composition. Scleraxis function is therefore essential for normal craniofacial form and function and vital for fish development.-Kague, E., Hughes, S. M., Lawrence, E. A., Cross, S., Martin-Silverstone, E., Hammond, C. L., Hinits, Y. Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.

Myogenin promotes myocyte fusion to balance fibre number and size.

Each skeletal muscle acquires its unique size before birth, when terminally differentiating myocytes fuse to form a defined number of multinucleated myofibres. Although mice in which the transcription factor Myogenin is mutated lack most myogenesis and die perinatally, a specific cell biological role for Myogenin has remained elusive. Here we report that loss of function of zebrafish myog prevents formation of almost all multinucleated muscle fibres. A second, Myogenin-independent, fusion pathway in the deep myotome requires Hedgehog signalling. Lack of Myogenin does not prevent terminal differentiation; the smaller myotome has a normal number of myocytes forming more mononuclear, thin, albeit functional, fast muscle fibres. Mechanistically, Myogenin binds to the myomaker promoter and is required for expression of myomaker and other genes essential for myocyte fusion. Adult myog mutants display reduced muscle mass, decreased fibre size and nucleation. Adult-derived myog mutant myocytes show persistent defective fusion ex vivo. Myogenin is therefore essential for muscle homeostasis, regulating myocyte fusion to determine both muscle fibre number and size.

Mef2 and the skeletal muscle differentiation program.

Mef2 is a conserved and significant transcription factor in the control of muscle gene expression. In cell culture Mef2 synergises with MyoD-family members in the activation of gene expression and in the conversion of fibroblasts into myoblasts. Amongst its in vivo roles, Mef2 is required for both Drosophila muscle development and mammalian muscle regeneration. Mef2 has functions in other cell-types too, but this review focuses on skeletal muscle and surveys key findings on Mef2 from its discovery, shortly after that of MyoD, up to the present day. In particular, in vivo functions, underpinning mechanisms and areas of uncertainty are highlighted. We describe how Mef2 sits at a nexus in the gene expression network that controls the muscle differentiation program, and how Mef2 activity must be regulated in time and space to orchestrate specific outputs within the different aspects of muscle development. A theme that emerges is that there is much to be learnt about the different Mef2 proteins (from different paralogous genes, spliced transcripts and species) and how the activity of these proteins is controlled.

Myotome adaptability confers developmental robustness to somitic myogenesis in response to fibre number alteration.

Balancing the number of stem cells and their progeny is crucial for tissue development and repair. Here we examine how cell numbers and overall muscle size are tightly regulated during zebrafish somitic muscle development. Muscle stem/precursor cell (MPCs) expressing Pax7 are initially located in the dermomyotome (DM) external cell layer, adopt a highly stereotypical distribution and thereafter a proportion of MPCs migrate into the myotome. Regional variations in the proliferation and terminal differentiation of MPCs contribute to growth of the myotome. To probe the robustness of muscle size control and spatiotemporal regulation of MPCs, we compared the behaviour of wild type (wt) MPCs with those in mutant zebrafish that lack the muscle regulatory factor Myod. Myodfh261 mutants form one third fewer multinucleate fast muscle fibres than wt and show a significant expansion of the Pax7+ MPC population in the DM. Subsequently, myodfh261 mutant fibres generate more cytoplasm per nucleus, leading to recovery of muscle bulk. In addition, relative to wt siblings, there is an increased number of MPCs in myodfh261 mutants and these migrate prematurely into the myotome, differentiate and contribute to the hypertrophy of existing fibres. Thus, homeostatic reduction of the excess MPCs returns their number to normal levels, but fibre numbers remain low. The GSK3 antagonist BIO prevents MPC migration into the deep myotome, suggesting that canonical Wnt pathway activation maintains the DM in zebrafish, as in amniotes. BIO does not, however, block recovery of the myodfh261 mutant myotome, indicating that homeostasis acts on fibre intrinsic growth to maintain muscle bulk. The findings suggest the existence of a critical window for early fast fibre formation followed by a period in which homeostatic mechanisms regulate myotome growth by controlling fibre size. The feedback controls we reveal in muscle help explain the extremely precise grading of myotome size along the body axis irrespective of fish size, nutrition and genetic variation and may form a paradigm for wider matching of organ size.